EP1739188B1 - Verfahren zur messung der aktivität von homocystein-thiolacton-hydrolase - Google Patents

Verfahren zur messung der aktivität von homocystein-thiolacton-hydrolase Download PDF

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EP1739188B1
EP1739188B1 EP05730497A EP05730497A EP1739188B1 EP 1739188 B1 EP1739188 B1 EP 1739188B1 EP 05730497 A EP05730497 A EP 05730497A EP 05730497 A EP05730497 A EP 05730497A EP 1739188 B1 EP1739188 B1 EP 1739188B1
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activity
htl
measurement
hydrolyzing enzyme
reagent
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EP1739188A4 (de
EP1739188A1 (de
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Masahiro Yamaguchi
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Alfresa Pharma Corp
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Alfresa Pharma Corp
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase

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  • the present invention relates to a method for measurement of the activity of the homocysteinethiolactone hydrolyzing enzyme, a reagent for measurement of the activity of the homocysteinethiolactone hydrolyzing enzyme, and a kit utilizing the reagent for measurement of the activity of the homocysteinethiolactone hydrolyzing enzyme.
  • Homocysteinemia is known to be one of risk factors of the development of myocardial infarction and arteriosclerosis. Precise mechanisms involved in it, however, have not yet been clarified, though such different hypotheses are proposed that are based on oxidation stress of homocysteine, impaired functioning of nitrogen oxide (NO), the endoplasmic reticulum put under stress, etc. Among these is the homocysteinethiolactone (hereinafter referred to as "HTL”) based hypothesis.
  • NO nitrogen oxide
  • HTL homocysteinethiolactone
  • Soukharev S. and Hammond D. disclose an assay for detection of organophasphatase activity using DEPFMU as a substrate.
  • paraoxonase enzymes and substrates thereof The activity of paraoxonase enzymes and substrates thereof is described by Billecke et al. ("Human Serum Paraoxonase (PON1) Isozymes Q and R Hydrolyze Lactones and Cyclic Carbonate Esters", Drug metabolism and disposition 28, 2000, 1335 - 1342 ).
  • HTL In the process of protein synthesis, certain aminoacyl-tRNAs are known to erroneously take up homocysteine instead of other amino acids. Although homocysteine thus taken up will not be incorporated into the protein being newly synthesized, cyclic HTL is formed in a process to correct this error. HTL freely passes through the cell membrane under a physiological condition and thus diffuses out of the cell. Chemically, HTL is rather active and acylates amino groups in proteins. In fact, it has been reported that HTL deactivates lysine oxidizing enzymes and modifies apoprotein B in the LDL, and some researcher think that HTL is the very culprit of the cell toxicity observed with homocysteine. On the other hand, since HTL, when added to the serum, is converted to homocysteine in a short time, it has been thought that a HTL hydrolyzing enzyme is present in the serum, protecting the body through decomposition of HTL.
  • PON a calcium-dependent enzyme
  • This enzyme was first found as an enzyme that hydrolyses paraoxon, a metabolite of parathion, a pesticide.
  • This enzyme hydrolyses other substrates such as phenyl acetate, too.
  • PON is thought to act in the direction to protecting serum lipoproteins from their oxidative degradation.
  • PON gene been shown to consist of at least three relating genes, PON1, PON2 and PON3.
  • PON and the HTL hydrolyzing enzyme is one and identical enzyme, no proper assessment of the enzyme in the body has been made so far. That is, it is unlikely that paraoxon, the substrate used in the measurement of PON activity, is the physiological substrate for the enzyme. Further, if there exists a PON2 that apparently is incapable of hydrolyzing paraoxon or a PON3 whose properties are not well known, there then would be a possibility that measurement of PON activity, which relies on paraoxon as the substrate, cannot serve as an accurate method of assessment. Recently, PON3 was purified from rabbit serum. Rabbit PON3 was found to be a protein having a molecular weight 40-KDa contained in the HDL fraction of the serum.
  • PON3 In contrast to PON1, PON3 has no paraoxon hydrolyzing activity, but hydrolyses lactones. And it has also been reported to function more protectively against LDL oxidation (see Non-patent Document 2). Further, it has been reported that PON1 also hydrolyzes various lactones (Non-patent Document 3).
  • HTL which is seemed to be a more physiological substrate, instead of the non-physiological substrate paraoxon, will lead to clarification of the precise role of the enzyme in the body and further to the possibility of revelation of the relations between the enzyme and arteriosclerotic diseases.
  • HTL hydrolyzing activity has so far been performed using HTL labeled with a radioisotope, [ 35 S].
  • this method of measurement entails difficult problems that prevent it from becoming a commonly used testing method, since it is subject to certain restrictions on operators and facilities in its practice, raises problems of safety and on how to dispose of the radioactive reagents, and, further, requires complicated processes such as thin layer chromatography to separate the reaction product, i.e., homocysteine, from undecomposed HTL.
  • human serum PON1 is known to hydrolyzes a number of lactones and thiolactones including paraoxon, phenyl acetate, 2-coumarone, dihydrocoumarin, homogentisic acid lactone, ⁇ -butyrolactone and derivatives thereof, ⁇ -angelicolactone, oxabicyclooctenones, 6-hydroxybutyrolactone, ⁇ -lactones such as ⁇ -valerolactone, ⁇ -caprolactone, ⁇ -thiobutyrolactone, homocysteinethiolactone, N-acetylhomocysteinethiolactone, propylene carbonate, 4-(1-propenyloxymethyl)-1,3-dioxolan-2-one, and the like (see above-cited Non-patent Document 3).
  • the objective of the present invention is to provide, in the field of measurement of HTL hydrolyzing enzyme activity, a method for the measurement that does not use a radioactive substrate, and is safe, easy to operate, and unsusceptible to other compounds occurring in a sample from the body.
  • HTL rapidly undergoes autolysis in near neutral solutions. Therefore, there has been a problem, in HTL-based measurement of the activity of the HTL hydrolyzing enzyme, that it gives high reagent-blank values and thus could affect the accuracy of measurement and stability of the solution used in it.
  • TBL ⁇ -thiobutyrolactone
  • the present inventor found the following advantages of the method for measurement of the activity of the HTL hydrolyzing enzyme using TBL as the substrate: that it is not affected by the presence of albumin (see the section of Examples); that the substrate is stable and thus gives lowered reagent-blank values (see the section of Examples); that its working solution is stored for an extended period of time; and that as it has no optical isomer, TBL exhibits steady specificity and is available at low cost.
  • the present inventor completed the present invention upon the finding that the activity of the HTL hydrolyzing enzyme can be measured with increased accuracy and specificity, without using a radioactive substrate, and more safely and with ease, and free of the influence of other compounds occurring in the body (albumin) by conducting a reaction using TBL as the substrate in the presence of a cholinesterase inhibitor.
  • the present invention provides a method for measurement of the activity of the homocysteinethiolactone hydrolyzing enzyme in a sample comprising mixing and reacting ⁇ -thiobutyrolactone and the sample in the presence of a cholinesterase inhibitor, and measuring 4-mercaptobutyric acid thereby produced.
  • the present invention further provides the method for measurement as mentioned above, wherein the reaction is conducted in the presence of a divalent cation.
  • a divalent cation Ca 2+ , Ni 2+ and Fe 2+ , for example, may be employed at a concentration of, e.g., 0.1-10 mM.
  • physostigmine or salts thereof, neostigmine or salts thereof, and 4-bromobenzeneboronic acid or salts thereof and the like may be used as a cholinesterase inhibitor.
  • physostigmine or salts thereof may be used at a concentration of, e.g., 10 ⁇ M to 10 mM, neostigmine or salts thereof, e.g., 10 ⁇ M to 10 mM, and 4-bromobenzeneboronic acid or salts thereof, e.g., 10 ⁇ M to 1 mM.
  • the present invention further provides the method for measurement as mentioned above, wherein the produced 4-mercaptobutyric acid is measured using a thiol group detection reagent.
  • a thiol group detection reagent 5,5'-dithiobis(2-nitrobenzoic acid), 4,4'-bis(dimethylamino)benzhidrol, 2,2'-dithiodipyridine, 4,4'-dithiodipyridine, 2,2'-dithiobis(5-nitropyridine) or 6,6'-dithiobisnicotinic acid, for example, may be used.
  • the present invention further provides a reagent for measurement of the activity of the homocysteinethiolactone hydrolyzing enzyme comprising ⁇ -thiobutyrolactone and a cholinesterase inhibitor.
  • reagent as a cholinesterase inhibitor, physostigmine or salts thereof, neostigmine or salts thereof, 4-bromobenzeneboronic acid or salt thereof, for example may be used.
  • the reagent may further comprise a thiol group detection reagent.
  • thiol group detection reagents 5,5'-dithiobis(2-nitrobenzoic acid), 4,4'-bis(dimethylamino)benzhidrol, 2,2'-dithiodipyridine, 4,4'-dithiodipyridine, 2,2'-dithiobis(5-nitropyridine) or 6,6'-dithiobisnicotinic acid, for example, may be used.
  • the present invention further provides a kit for measurement of the activity of the homocysteinethiolactone hydrolyzing enzyme.
  • a kit may comprise, e.g., containers separately containing ⁇ -thiobutyrolactone and a cholinesterase inhibitor, or further comprise an additional container separately containing a thiol group detection reagent.
  • it may comprise multiple containers or one container having multiple separated zones, containing a mixture consisting of ⁇ -thiobutyrolactone and a cholinesterase inhibitor or further of a thiol group detection reagent, or comprise strips of paper impregnated with ⁇ -thiobutyrolactone and a cholinesterase inhibitor or further with a thiol group detection reagent.
  • the present invention as defined above provides a means for measurement of the activity of the homocysteinethiolactone hydrolyzing enzyme, which means is safe, quick, highly specific and convenient to use in daily examinations, without using a radioactive substrate. Furthermore, the present invention is particularly useful in the measurement of the activity of the homocysteinethiolactone hydrolyzing enzyme in samples taken from the body, such as the blood, serum, plasma and the like, and thereby makes it easy to give diagnosis of, and comprehend the progression of, diseases resulting from alterations of HTL hydrolyzing enzyme activity.
  • the first characteristic of the method for the measurement of the activity of the HTL hydrolyzing enzyme consists in that it utilizes TBL, in place of HTL, as a substrate, which is reacted with a HTL hydrolyzing enzyme-containing sample, and 4-mercaptobutyric acid thus formed through decomposition by the HTL hydrolyzing enzyme is measured to determine the activity of the HTL hydrolyzing enzyme.
  • the second characteristic of the present invention consists in that the reaction of TBL with HTL hydrolyzing enzyme in a sample is conducted in the presence of a cholinesterase inhibitor.
  • the third characteristic of the present invention consists in that it measures 4-mercaptobutyric acid produced by the reaction, based on the change in the color development by reacting it with a SH detection reagent. According to the present invention, the measurement can be performed safely, quickly, specifically and conveniently, for it does no use a radioactive substrate.
  • the reagent for measurement of the activity of the HTL hydrolyzing enzyme of the present invention comprises a substrate for HTL hydrolyzing enzyme.
  • a substrate for HTL hydrolyzing enzyme.
  • TBL is employed as a substrate.
  • the substrate is provided in the form of a liquid.
  • the substrate is provided at a concentration that allows its final concentration in the reaction solution to fall within the range of 5-100 mM, more preferably 10-50 mM.
  • the reagent for measurement of the activity of the HTL hydrolyzing enzyme is preferably provided in the form of a liquid.
  • the pH of a reaction solution of HTL hydrolyzing enzyme is about 6-9, preferably 7-8, and the reagent for measurement of the activity of the HTL hydrolyzing enzyme may contain, if needed, a buffering agent to make the pH of the reaction solution fall within the region, in accordance with the nature of the sample to be analyzed.
  • the substrate for HTL hydrolyzing enzyme may be dissolved in water or a buffer of pH 2-9 (e.g., HEPES buffer, citrate buffer, tartrate buffer, acetate buffer, Tris buffer, borate buffer, MOPS buffer, PIPES buffer, Good's buffer, etc.).
  • a buffer of pH 2-9 e.g., HEPES buffer, citrate buffer, tartrate buffer, acetate buffer, Tris buffer, borate buffer, MOPS buffer, PIPES buffer, Good's buffer, etc.
  • a SH detection reagent may be dissolved in a buffer of pH 2-9 (e.g., HEPES buffer, citrate buffer, tartrate buffer, acetate buffer, Tris buffer, borate buffer, MOPS buffer, PIPES buffer, Good's buffer, etc.).
  • a buffer of pH 2-9 e.g., HEPES buffer, citrate buffer, tartrate buffer, acetate buffer, Tris buffer, borate buffer, MOPS buffer, PIPES buffer, Good's buffer, etc.
  • Preferable pH is 6-8.
  • a SH detection reagent may be any one of the compounds, without specific limitation, insofar as it, after reacting with a SH group, quantitatively exhibits color changes (in the visible or ultraviolet range).
  • Examples include DTNB (5,5'-dithiobis(2-nitrobenzoic acid)), 4,4'-bis(dimethylamino)benzhidrol, 2,2'-dithiodipyridine, 4,4'-dithiodipyridine, 2,2'-dithiobis(5-nitropyridine) or 6,6'-dithiobisnicotinic acid, for example, may be used.
  • a SH detection reagent may be used at its final concentration falling within the range of 0.1-10 mM, more preferably 0.5-2 mM in the reaction solution.
  • a cholinesterase inhibiter may be any one of the compounds which specifically inhibit cholinesterase, and such cholinesterase inhibitors may be used as physostigmine and salts thereof (e.g., physostigmine sulfate), neostigmine or salts thereof (e.g., neostigmine bromide), 4-bromobenzeneboronic acid or salts thereof, tetraethyl pyrophosphate, tri-o-cresyl phosphate, octamethylpyrophosphophole tetramide, tetramonoisopropylphosphole tetramide, phosphorin and salts thereof (e.g., phosphorin iodide), edrophonium and salts thereof (e.g.
  • edrophonium bromide ethopropazine and salts thereof (e.g., ethopropazine hydrochloride), fluostigmine, tetrazoline and salts thereof (e.g., tetrazoline hydrochloride), tetrazolone and salts thereof (e.g., tetrazolone hydrochloride), gramine, desoxypeganine and salts thereof (desoxypeganine hydrochloride) and the like.
  • physostigmine and salts thereof neostigmine and salts thereof, and 4-bromobenzeneboronic acid and salts thereof.
  • a choline esterase in use may be set at any concentration as desired, insofar as it, at the concentration, effectively inhibits cholinesterase in a given sample.
  • physostigmine or its salt, or neostigmine bromide or its salt they may be used at, e.g., 10 ⁇ M to 10 mM, and more preferably 20 ⁇ M to 5 mM.
  • 4-bromobenzeneboronic or salts thereof they may be used at, e.g., 10 ⁇ M to 1 mM, more preferably at 20 ⁇ M to 100 ⁇ M.
  • the reagent of the present invention for measurement of the activity of the HTL hydrolyzing enzyme simultaneously contains a divalent cation.
  • divalent cations include Ni 2+ , Fe 2+ , Ca 2+ etc., among which Ca 2+ is particularly preferred.
  • a divalent cation may be used at its final concentration falling within the range of 0.1-10 mM, more preferably 0.5-5 mM in the reaction solution.
  • the reagent of the present invention for measurement of the activity of the HTL hydrolyzing enzyme may contain a surfactant.
  • surfactants examples include, but not limited to, anionic surfactants, nonionic surfactants, bile salts and derivatives of bile salts, etc. It is possible to use a single type of surfactant or to use two or more types of surfactants in combination. Further, it is also possible to use two or more surfactants of the same type.
  • an anionic surfactant is preferably at least one compound selected from fatty acid salts (e.g., sodium stearate, potassium oleate, etc.), sulfuric acid alkyl ester salts (e.g., sodium lauryl sulfate, lithium lauryl sulfate, etc.), benzenesulfonic acid alkyl ester salts (e.g., sodium lauryl benzenesulfonate, sodium 4-n-octyl benzenesulfonate, etc.), naphthalenesulfonic acid alkyl ester salts (e.g., sodium 2-naphthalenesulfonate), sulfosuccinic acid alkyl ester salts (e.g., sodium dialkyl sulfosuccinate), alkyl diphenylether disulfonic acid ester salts (e.g., sodium alkyl diphenyl ether dis
  • a nonionic surfactant is at lease one compound preferably selected from polyoxyethylene alkyl ethers (e.g., polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene stearyl ether, polyoxyethylene oleyl ether, etc.), polyoxyethylene alkylaryl ethers (e.g., polyoxyethylene octylphenyl ether, polyoxyethylene nonylphenyl ether, polyoxyethylene isooctylphenyl ether, etc.), polyoxyethylene derivatives (polyoxyethylene polyoxypropylene condensate, etc.), sorbitan fatty acid esters (e.g., sorbitan monolaureate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleate, sorbitan distearate, etc.
  • bile acid salts or bile acid derivatives is preferably at least one compound selected from sodium cholate, sodium deoxycholate, sodium chenodeoxycholate, sodium dehydrocholate, sodium taurocholate, sodium taurolitocholate, sodium taurodeoxycholate, sodium taurochenodeoxycholate, sodium tauroursodeoxycholate, sodium taurodehydrocholate, 3-[(3-chloramidepropyl)dimethylammonio]propanesulfonic acid (CHAPS), 3-[(3-chloramidepropyl)dimethylammonio]-2-hydroxypropanesulfonic acid (CHAPSO), N,N-bis-(3-D-gluconamidepropyl)cholamide (BIGCHAP), and N,N-bis(3-D-gluconamide-propyl)deoxycholamide (deoxy-BIGCHAP).
  • the reagent of the present invention for measurement of the activity of the HTL hydrolyzing enzyme may contain, in addition to those above-mentioned, one or more compounds conventionally used in enzyme activity measurement, such as buffering agents, stabilizers, activators, diluents, preservatives, etc.
  • 4-mercaptobutyric acid which is formed by hydrolysis of TBL by HTL hydrolyzing enzyme in the presence of a cholinesterase inhibitor, may be measured by any one of methods as desired, such as HPLC or the like.
  • preferred in the present invention is a method in which 4-mercaptobutyric formed by the reaction is measured, qualitatively or quantitatively, by reacting it with a SH-group detection reagent and measuring thus caused changes in color.
  • the concentration of a SH-group detection reagent in the reaction liquid may be set as desired at any one of concentrations conventionally employed for SH-group detection, e.g., 0.1-10 mM, or 0.5-5 mM.
  • a sample with which the activity of the HTL hydrolyzing enzyme is to be measured may be human or animal body fluid such as blood, serum, urine or amnion liquid, as well as human or animal cells, organs or their extract liquid and the like.
  • a HTL hydrolyzing enzyme product and a sample such as serum to be analyzed containing the HTL hydrolyzing enzyme are subjected to the reaction for measurement after dilution as desired with a buffer of pH 6-9 (e.g., HEPES buffer, Tris buffer, phosphate buffer, borate buffer, MOPS buffer, PIPES buffer Good's buffer, etc.) and kept at 20-40 °C.
  • a buffer of pH 6-9 e.g., HEPES buffer, Tris buffer, phosphate buffer, borate buffer, MOPS buffer, PIPES buffer Good's buffer, etc.
  • Sodium chloride and potassium chloride may be added to these buffers to attain a proper concentration of salts.
  • absorbance is measured at a wavelength that is suitable to the reagent employed. Measurement may be made either by an end-point method or a rate method. In the case of an end-point method, it is necessary that, for determining the activity of the HTL hydrolyzing enzyme, absorbance be subtracted that is generated as a result of color development by the reaction of the SH-group detection reagent and free SH-groups of proteins occurring in a sample, by reacting the sample with a reagent for measurement which contains other ingredients but the substrate. In the case of a rate method, measurement of absorbance may be made while the change in color development is taking place quantitatively. HTL hydrolyzing enzyme activity may be calculated on the basis of the molecular extinction coefficient of the SH group detection reagent when it develops color, or based on absorbance measured after color development using a predetermined quantity of a standard compound which has free SH groups.
  • Measurement of the activity of the HTL hydrolyzing enzyme according to the present invention may be carried out either by hand or on an automatic analyzer. Change in color is measured which is generated by the reaction of free SH group-carrying 4-mercaptobutyric acid formed by HTL hydrolyzing enzyme and a SH-group detection reagent.
  • HTL hydrolyzing enzyme activity can be determined with a high degree of accuracy by detecting the change in absorbance at the wavelength of 400-500 nm and then calculating the change in absorbance per unit of time (so-called rate assay method).
  • a SH-group detection solution prepared by dissolving a reagent in a suitable buffer solution (for example, 0.2 M MOPS buffer (pH 7.2) containing SH-group detection reagent, 0.1 mM physostigmine sulfate, and 5 mM CaCl 2 ) and a substrate solution are prepared.
  • a suitable buffer solution for example, 0.2 M MOPS buffer (pH 7.2) containing SH-group detection reagent, 0.1 mM physostigmine sulfate, and 5 mM CaCl 2
  • a suitable amount of human pool serum or human serum is mixed with the SH-group detection solution, and the substrate solution then is added to the mixture to react.
  • Reaction temperature may be 20-40 °C, preferably 37°C.
  • absorbance is measured in a suitable time window (e.g., from 2-minute up to 4-minute time points), change in absorbance per unit of time calculated, and from this, based on the molecular extinction coefficient of the SH detection reagent, the activity value of the HTL hydrolyzing enzyme may be determined.
  • a suitable time window e.g., from 2-minute up to 4-minute time points
  • the aforementioned SH-group detection solution and a substrate solution are first prepared. Using, for example, a H-7170 automatic analyzer (Hitachi, Ltd.), measurement is made in accordance with parameters for the measurement. Human pool serum or human serum is mixed with the SH-group detection solution, and, after a predetermined length of time, the substrate solution is further mixed to allow the reaction to start. Change in absorbance (time profile) then is monitored, and amount of the change in absorbance per unit of time (1 min) is determined from 2-minute time point after the start of the reaction up to 4-minute time point.
  • H-7170 automatic analyzer Hitachi, Ltd.
  • the activity value of HTL hydrolyzing enzyme for each sample is calculated according to the following mathematical formula, based on the change in absorbance per unit of time measured with the sample ( ⁇ ES), the change in absorbance per unit of time measured with purified water in place of the sample ( ⁇ EB), as well as the molecular extinction coefficient of the SH-group detection reagent ( ⁇ 1) in the reaction solution and at the main wavelength for the measurement, and the molecular extinction coefficient of the SH-group detection reagent ( ⁇ 2) at the supplementary wavelength for measurement.
  • the kit of the present invention for measurement of the activity of the HTL hydrolyzing enzyme comprises the above-mentioned reagent for measurement of the activity of the HTL hydrolyzing enzyme which contains a substrate, a SH-group detection reagent, a divalent cation, a cholinesterase inhibitor, and, as needed, other additives.
  • the kit may be provided, for example, in the form of ampoules or vials each containing TBL, a cholinesterase inhibitor, or a SH-group detection reagent, or a mixture of them, or in the form of wells in each of which a predetermined amount of the reagent for measurement of the activity of the HTL hydrolyzing enzyme is injected, or in the form comprising a container containing the reagent for measurement of the activity of the HTL hydrolyzing enzyme and wells in which the measurement is to be made.
  • a sample e.g., serum
  • the kit of the present invention for the measurement of the activity of the HTL hydrolyzing enzyme may be in the form of testing strips of, e.g., filter paper impregnated with the above-mentioned reagent for measurement of the activity of the HTL hydrolyzing enzyme containing the substrate TBL, a cholinesterase inhibitor, and, as desired, a SH-group detection reagent and a divalent cation, and, as needed, other additives.
  • testing strips may be prepared by soaking those strips in a solution of the reagent and then drying them. By dipping a testing strip in serum and the like, HTL hydrolyzing enzyme activity contained is qualitatively detected.
  • a SH-group detecting reagent is provided in a separately attached container, from which it is dropped onto a testing strip in which a hydrolysis reaction has taken place. Even with these testing strips-type kits, activity can, though roughly, be determined using a color table for comparison. Therefore, the kit may contain such a table for comparison.
  • the kit of the present invention may be provided in such a form that a solution containing the substrate and a solution containing a SH-group detection reagent are respectively contained in separate containers and mixed when they are used.
  • the substrate may be provided as a solution in water, a buffer solution or in a solvent such as alcohol (methanol, ethanol, etc.) or dioxane, or, alternatively, a solution and/or solvent for dissolving the substrate may separately be provided in discrete containers.
  • a SH-group detection reagent may be provided as a solution in water, a buffer solution or in a solvent such as alcohol (methanol, ethanol, etc.), N,N-dimethylformamide, acetone and the like, or, alternatively, a solution and/or solvent for dissolving the SH-group detection reagent may separately be provided in separate containers.
  • a solvent such as alcohol (methanol, ethanol, etc.), N,N-dimethylformamide, acetone and the like
  • a solution and/or solvent for dissolving the SH-group detection reagent may separately be provided in separate containers.
  • a solution and/or solvent for dissolving the SH-group detection reagent may separately be provided in separate containers.
  • those forms may be selected as desired, considering easiness of operation, amount to be used, etc.
  • the present invention is completed on the basis of novel finding that TBL does not serve as substrate for the esterase-like activity of albumin but do serve as a substrate for cholinesterase, that
  • TBL TBL-like compounds that does not serve as substrates for the esterase-like activity of albumin but substrates for cholinesterase, will fall within the scope of equivalents to the present invention.
  • the fraction containing chylomicron, VLDL and LDL and the fraction containing HDL, and human plasma (heparinized plasma) were dialyzed against 0.1 M HEPES buffer containing 0.1 mM CaCl 2 (pH 7.6) and was made samples.
  • Human albumin (A-1887, Sigma), human albumin (A-3782, Sigma), human albumin (05418, Fluca), human albumin (126654, Calbiochem), human albumin (A-1653, Sigma) were dissolved in purified water to prepare 5 % human albumin solutions (hereinafter referred to as "HSA").
  • Lipidserum II (Eiken Chemical Co., Ltd) was dissolved in 3 ml of purified water. Determiner Standard HDL-C.LDL-C for measurement (Kyowa Medex Co., Ltd.) was dissolved in 1 ml of purified water. Five ml of human serum was collected using Veneject II vacuum blood sampling tube, plain (Terumo Corporation).
  • Hydrolyzing enzyme activity for HTL was measured with purified water and above-mentioned samples. Following solutions were prepared:
  • H-7170 automatic analyzer (Hitachi, Ltd.) was used. Parameters for measurement were as follows.
  • HTL hydrolyzing enzyme activity was found localized in the HDL fraction. And, it was confirmed that, the activity of the HTL hydrolyzing enzyme does not undergo modification in the presence of a cholinesterase inhibitor, and that HTL is not hydrolyzed by purified human cholinesterase. Further, the hydrolyzing activity exhibited by HSA for the substrate HTL was found to be calcium-independent. Therefore, the hydrolyzing activity is considered to be attributable to HSA itself.
  • H-7170 automatic analyzer (Hitachi, Ltd.) was used. Parameters for measurement were as follows.
  • HTL hydrolyzing enzyme activity for TBL was found localized in the HDL fraction. And, it was confirmed that TBL serves as a substrate for cholinesterase, for the hydrolyzing enzyme activity for TBL is greatly altered in the presence of a cholinesterase inhibitor, and purified human cholinesterase exhibits hydrolyzing enzyme activity for TBL. Of five different HSAs, hydrolyzing enzyme activity for TBL was observed only with A-1653. As the hydrolyzing enzyme activity for TBL with A-1653 was eliminated by a cholinesterase inhibitor, it was considered due to contamination with cholinesterase.
  • Cholinesterase activity was measured with the above-mentioned samples and unprepared serum. Measurement of cholinesterase activity was carried out using Nescoat ChE V-2 (Alfresa Pharma Coporation) according to the package insert.
  • Fig. 5 shows the correlation between the results (Y) of measurement of the hydrolyzing enzyme activity for HTL in the presence of calcium (indicated as HTLase in the figure) and the results (X) of measurement of the hydrolyzing activity for TBL in the absence of a cholinesterase inhibitor (indicated as TBLase in the figure).
  • Fig. 6 shows the correlation between the calcium dependent part of activity (Y) of the HTL hydrolyzing enzyme activity (which is the potion of the activity enhanced by calcium ion, i.e., the portion corresponding to the net HTL hydrolyzing activity of interest) and the results (X) of measurement of the TBL hydrolyzing enzyme activity in the absence of a cholinesterase inhibitor.
  • Fig. 7 shows the correlation between the calcium dependent part of activity (Y) of the HTL hydrolyzing enzyme activity and the calcium dependent part of activity (X) of the TBL hydrolyzing enzyme activity.
  • Fig. 8 shows the correlation between the calcium dependent part of activity (Y) of the HTL hydrolyzing enzyme activity and the results of measurement of TBL hydrolyzing enzyme activity (X) in the presence of a cholinesterase inhibitor.
  • the correlation ( Fig. 8 ) between the TBL hydrolyzing enzyme activity in the presence of a cholinesterase inhibitor and the calcium dependent part of activity of the HTL hydrolyzing enzyme activity is equivalent to the correlation ( Fig. 7 ) between the calcium dependent part of activity of the TBL hydrolyzing enzyme activity and the calcium dependent part of activity of the HTL hydrolyzing enzyme activity. Therefore, TBL hydrolyzing enzyme activity in the presence of a cholinesterase inhibitor strongly reflects the net HTL hydrolyzing enzyme activity.
  • the method of the present invention for measurement of the activity of the HTL hydrolyzing enzyme can be used as a safe, quick and convenient method for measurement in daily testing, for it makes it possible to measure the activity of the HTL hydrolyzing enzyme in a sample without employing a radio-labeled substrate.
  • the method of the present invention for measurement of the activity of the HTL hydrolyzing enzyme allows diagnosis and prognosis of relating diseases to be done within a short time, for it has such advantages over conventional methods that it can shorten the time required for measurement and is applicable to automatic analyzers, and thus enables more accurate and quicker measurement as compared with conventional methods. Therefore, the present invention provides a highly useful method for measurement of the activity of the HTL hydrolyzing enzyme.

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Claims (13)

  1. Verfahren zur Messung der Aktivität eines Homocystein-Thiolacton hydrolisierenden Enzyms in einer Probe, wobei die Methode ein Mischen und Reagieren von γ-Thiobutyrolacton und der Probe in der Gegenwart eines Cholinesterase-Inhibitors, und Messung der dadurch hergestellen 4-Mercaptobutansäure umfasst.
  2. Verfahren gemäß Anspruch 1, wobei die Reaktion durchgeführt wird in der Gegenwart eines zweiwertigen Kations.
  3. Verfahren gemäß Anspruch 1 oder 2, wobei das zweiwertige Kation ausgewählt ist aus der Gruppe, bestehend aus Ca2+, Ni2+ und Fe2+.
  4. Verfahren gemäß einem der Ansprüche 1 bis 3, wobei die Konzentration des zweiwertigen Kations 0,1 - 10 mM ist.
  5. Verfahren gemäß einem der Ansprüche 1 bis 4, wobei der Cholinesterase-Inhibitor ausgewählt ist aus der Gruppe, bestehend aus Physostigmin und Salzen davon, Neostigmin und Salzen davon, und 4-Brombenzolborsäure und Salzen davon.
  6. Verfahren gemäß Anspruch 5, wobei die Konzentration von Physostigmin oder Salzen davon 10 µM bis 10 mM ist, die Konzentration von Neostigmin oder Salzen davon 10 µM bis 10 mM ist, und die Konzentration von 4-Brombenzolborsäure oder Salzen davon 10 µM bis 1 mM ist.
  7. Verfahren gemäß einem der Ansprüche 1 bis 6, wobei die hergestellte 4-Mercaptobutansäure gemessen wird unter Verwendung eines Nachweisreagenz für Thiolgruppen.
  8. Verfahren gemäß Anspruch 7, wobei das Nachweisreagenz für Thiolgruppen ausgewählt ist aus der Gruppe, bestehend aus 5,5'-Dithiobis(2-nitrobenzoesäure), 4,4'-Bis-(dimethylamino)benzhydrol, 2,2'-Dithiodipyridin, 4,4'-Dithiodipyridin, 2,2'-Dithiobis-(5-nitropyridin) und 6,6'-Dithiobisnicotinsäure.
  9. Reagenz zur Messung der Aktivität eines Homocystein-Thiolacton hydrolisierenden Enzyms umfassend γ-Thiobutyrolacton und einen Cholinesterase-Inhibitor.
  10. Reagenz gemäß Anspruch 9, weiter umfassend ein Nachweisreagenz für Thiolgruppen.
  11. Kit zur Messung der Aktivität eines Homocystein-Thiolacton hydrolisierenden Enzyms, wobei das Kit Container umfasst, die getrennt γ-Thiobutyrolacton und einem Cholinesterase-Inhibitor beinhalten, oder weiter einen weiteren Container umfasst, der separat eine Nachweisreagenz für Thiolgruppen beinhaltet.
  12. Kit zur Messung der Aktivität eines Homocystein-Thiolacton hydrolisierenden Enzyms, wobei das Kit mehrere Container oder einen Container mit mehreren getrennten Zonen, beinhaltend eine Mischung bestehend aus γ-Thiobutyrolacton und einem Cholinesterase-Inhibitor oder weiter eines Nachweisreagenz für Thiolgruppen umfasst.
  13. Kit zur Messung der Aktivität eines Homocystein-Thiolacton hydrolisierenden Enzyms, wobei das Kit Streifen umfasst, die mit γ-Thiobutyrolacton und einem Cholinesterase-Inhibitor imprägniert sind oder weiter mit einem Nachweisreagenz für Thiolgruppen.
EP05730497A 2004-04-19 2005-04-12 Verfahren zur messung der aktivität von homocystein-thiolacton-hydrolase Ceased EP1739188B1 (de)

Applications Claiming Priority (2)

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JP2004122926A JP4729264B2 (ja) 2004-04-19 2004-04-19 ホモシステインチオラクトン加水分解酵素活性の測定法
PCT/JP2005/007050 WO2005103286A1 (ja) 2004-04-19 2005-04-12 ホモシステインチオラクトン加水分解酵素活性の測定法

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WO2010001598A1 (ja) * 2008-06-30 2010-01-07 積水メディカル株式会社 結合アッセイ用多孔性固相及びこれを用いた結合アッセイ法
JP5832995B2 (ja) * 2010-04-30 2015-12-16 協和メデックス株式会社 低密度リポ蛋白中のコレステロールの測定方法、測定用試薬及び測定用キット

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WO2005103286A1 (ja) 2005-11-03
JP2005304329A (ja) 2005-11-04
US20070243571A1 (en) 2007-10-18
EP1739188A1 (de) 2007-01-03
US7534581B2 (en) 2009-05-19
JP4729264B2 (ja) 2011-07-20

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